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Microspectrophotometry
Validation
Reasons for Changing
Instruments
•
Reduced reliability.
•
Limited efficiency.
•
Limited availability and cost of
replacement parts.
•
Problems with Service contract.
Validation Plan
• Once a new instrument has been
purchased it is necessary to produce a
validation plan.
• The plan will include the parameters to be
tested.
• Each parameter will, where possible, have
a standard and target:
• Standard – criteria that must be met for the
system to be considered acceptable.
• Target – performance criteria that are desirable
but not essential.
Measurement Parameters
• Before beginning the validation of the
instrument measurement parameters must
be decided.
• Use the parameters recommended by the
manufacturer or those which proved to be
most acceptable when preliminary
measurements are carried out during
installation or initial testing.
• Check the settings using calibration
standards or samples run on other
validated instruments.
Validation Plan Parameters
• The Instrument:
• Measurement Window – optimum size and
boundary accuracy.
• Wavelength Registration – accuracy of peak
position.
• Photometric Measurement – check intensity at
each wavelength.
• Noise Level – check 100% line.
• Second Order Effects – this is where the
measurement at a given wavelength includes
some component of light from multiples of that
wavelength.
Validation Plan Parameters
continued
• The Instrument:
• Spectral Distortion – Caused by system overload
i.e. excess light reaching the detector.
• Focus Level – Is the level of focus critical to
spectral shape and peak position?
• Limit of Detection – The system should be able
to generate acceptable results from fibres over
an acceptable absorbance range e.g. 0.1 – 1.4AU
• Fluorescence – The system should be able to
measure fluorescent samples.
• Time – Is the system faster, with respect to
producing spectra, than your previous system?
Validation Plan Parameters
continued
• The Instrument:
• Operator Effects – Do different operators
generate identical data?
• Bleaching Effects – If fibres are
measured at the same point during
Validation will they bleach?
• External Interference – Does the
laboratory environment influence the
results e.g. lighting, heating, vibrations.
• Polarisation Effects – Does polarisation
affect the results?
Validation Plan Parameters
continued
• Software Issues: (both data capture and analysis).
The areas to be validated in this section will to some
degree depend on the software used and the needs of the
laboratory.
• Algorithms:
• Are the algorithms used to calculate absorbance
values accurate?
• The peak finding algorithm should contain no
logical flaws and the software must implement it
correctly.
• Colour Calculations – If complementary chromaticy
co-ordinates are going to be generated they must
be accurate.
Validation Plan Parameters
continued
• Software issues:
• Output Spectrum – does the output spectrum
accurately reflect the data in both intensity and
absorbance plots?
• Input of Case Data e.g. case number etc.– Is this
possible and to an acceptable level?
Validation Plan Parameters
continued
• Other:
• Consumable Variables e.g. glycerol, slides.
Validation Plan – An Example
• Wavelength Registration.
The Standards and Targets were set as follows:
Standard (1) – The wavelength must be accurate to +/2.5nm.
Target (1) – The wavelength must be accurate to +/- 1.0nm
Standard (2) – The adjusted root mean square standard
deviation of the mean of each peak must be less than
1.5nm.
Target (2) – The adjusted root mean square standard
deviation of the mean of each peak must be less than
1.0nm.
Validation Plan - An Example
continued
• Wavelength Registration.
Standard (3) – There must be less than 0.5nm drift in
the mean position over 24 hours.
Target (3) – There must be no shift in peak position
throughout the validation study.
A Holmium Oxide sample was measured on 8
occasions (10 measurements each time) over a 16
day period. Where necessary the results were
compared with published peak positions for
Holmium Oxide.
Validation Plan – An Example
continued
• Wavelength Registration.
Conclusions were as follows:
• The wavelength accuracy was within 1nm
(the Target).
• The wavelength precision fell within the
acceptable limits of 2.5nm; all bar one
peak was within the target range of 1.0nm.
• There was no evidence of a shift in peak
position throughout the study.
• All results demonstrated that they were
reproducible.